Polysaccharides containing α-1,4-glucan chains and method for producing same

ABSTRACT

The invention relates to a method for producing polysaccharides containing α-1,4-glucan chains. According to the inventive method, a glucosyl group acceptor undergoes a chain prolongation reaction by reacting it with saccharose in the presence of an amylosaccharase. The amount of the glucosyl group acceptor in the reaction mixture is chosen in such a way that the mole ratio of the available ends of the glucosyl group acceptor to the saccharose is at least 1:1,000 and/or the weight ratio of the glucosyl group acceptor to the saccharose is at least 1:0.

This is the U.S. national phase of International Application No.PCT/EP99/09299 filed Nov. 30, 1999, the entire disclosure of which isincorporated herein by reference.

DESCRIPTION

The present invention relates to α-1,4-glucan-chain-containingpolysaccharides and to a process for their preparation.

Polysaccharides are polymers which are composed of numerousglycosidically bound monosaccharides. Polysaccharides occur both inhigher organisms and in microorganisms such as bacteria and therefulfill, for example, the function of storage and framework substances.The polysaccharides are used commercially, inter alia, as aids andadditives in the food industry, in light industry, in health care and inanalysis.

Glucans are polysaccharides which solely consist of glucose monomers. Inthe α-1,4-glucans, these glucose radicals are linked to one another byα-1,4-glycosidic bonds. α-1,4-Glucans, owing to their physicochemicalproperties, can be used to produce films which are color-free, odorlessand tasteless, non-toxic and biodegradable. Already, there are numerousapplications for such films, for example in the food industry, thetextile industry and the glass fiber industry.

The most frequently occurring natural α-1,4-glucan is amylose, a starchconstituent. Amylose is already used to produce fibers whose propertiesresemble those of natural cellulose fibers and make possible theirpartial or complete replacement in paper making. In pharmacy, amylose isused as a filler for tablets, pastes and as additive to skin protectionsubstances. In the food industry, it serves as thickener and binder forpuddings, soups, sauces, mayonnaises, cream fillings and as a gelatinsubstitute. Amylose is also used as a binder in the production ofsound-insulating wall panels.

Amylopectin, the main constituent of starch, and glycogen are furtherpolysaccharides whose main chains consist of glucose radicals havingα-1,4-glycosidic linkage. These polysaccharides bear side chains whichare linked to the main chain via α-1,6-glycosidic bonds. Thesepolysaccharides are also used to a great extent in industry.

The isolation of α-1,4-glucans such as starch and glycogen from plantand animal organisms is complex and costly and does not always lead toproducts having reproducible properties. For this reason, bacteria whichcan produce such glucans have increasingly become the subject ofattention.

In most bacteria, polysaccharides are synthesized in a similar manner toin higher organisms, via nucleotide-activated sugars. Thus, in mostbacteria, the biosynthesis of glycogen involves three enzymes, that isto say ADP-glucose phosphorylase, which catalyzes the formation ofADP-glucose from glucose-1-phosphate and ATP, glycogen synthase whichtransfers the glucose from ADP-glucose to the growing glucan chain, anda branching enzyme which introduces α-1,6-links into the linearα-1,4-glucan chain. However, in some bacteria polysaccharide synthesiscan also take place without the participation of activated sugars.

One of the bacterial systems which is able to synthesize polysaccharideswithout the participation of nucleotide sugars has been found inbacteria of the genus Neisseria. In these bacteria, polysaccharideshaving a similar structure to glycogen are synthesized by the enzymeamylosucrase directly from sucrose, the natural substrate of the enzyme[Okada, G., and E. J. Hehre, J. Biol. Chem. 249:126-135 (1974);MacKenzie, C. R. et al., Can. J. Microbiol. 23:1303-1307 (1977);MacKenzie, C. R. et al., Can. J. Microbiol. 24:357-362 (1978)].

Amylosucrase (sucrose: 1,4-α-glucan 4-α-glucosyltransferase, E. C.2.4.1.4.) catalyzes the formation of α-1,4-glycosidically linkedglucans, by transferring the glucosyl radical of the sucrose molecule tothe growing polymer chain, with the release of D-fructose, according tothe following reaction equation

Sucrose+(α-1,4-D-glucosyl)_(n)→D-fructose+(α-1,4-D-glucosyl)_(n+1).

Nucleotide-activated sugars or cofactors are not required in thisreaction. However, the enzyme is stimulated by the presence of glucosylgroup acceptors (or primers), for example oligo- and polysaccharidessuch as amylose or glycogen, to which the glucosyl radical of thesucrose is transferred according to the above reaction equation withα-1,4-glucan chain extension [Okada, G., and E. J. Hehre, J. Biol. Chem.249:126-135 (1974); Remaud-Simeon, M. et al., In S. B. Petersen, B.Svenson and S. Pedersen (editors), Carbohydrate bioengineering, pp.313-320 (1995); Elsevier Science B. V., Amsterdam, Netherlands].

Amylosucrases have been found to date only in bacteria of the genusNeisseria. The enzyme which is expressed constitutively in the bacteria,is extremely stable and binds very firmly to its polymerization product.In most species investigated the enzyme is localized intracellularly,but in Neisseria polysaccharea, the amylosucrase is secreted. The genefor amylosucrase from Neisseria polysaccharea has in the interim beenisolated and expressed using genetic engineering methods. It has beenfound that the enzyme highly probably only catalyzes the formation oflinear α-1,4-glucan chains (WO 95/31553).

The use of amylosucrase from N. polysaccharea for preparing linearα-1,4-glucans has already been proposed in WO 95/31553. However, aproblem in the use of amylosucrases for producing polysaccharides isthat the polysaccharides usually formed in the presence of amylosucrasehave highly variable molecular weights, i.e. a high polydispersity orbroad molecular weight distribution. However, for an industrialapplication, because of their more homogeneous physicochemicalproperties, polysaccharide preparations having a molecular weight asuniform as possible, that is to say low polydispersity, are desired.

The object of the present invention was therefore to provideα-1,4-glucan-chain-containing polysaccharides having a lowpolydispersity.

This object was achieved by the processes and polysaccharides describedin the claims.

The present invention therefore relates to a process which comprises aglucosyl group acceptor being subjected to a chain extension reaction byreaction with sucrose in the presence of an amylosucrase, the amount ofglucosyl group acceptor in the reaction mixture being chosen so that themolar ratio of glucosyl group acceptor ends available for chainextension to sucrose is at least 1:1 000 and/or the weight ratio ofglucosyl group acceptor to sucrose is at least 1:50.

The invention also relates to a process which comprises subjecting aglucosyl group acceptor to a chain extension reaction by reaction withsucrose in the presence of an amylosucrase and with addition offructose.

α-1,4-Glucan-chain-containing polysaccharides which are available bythese processes are also subject-matter of this invention.

The inventively used glucosyl group acceptors are compounds on whichsynthesis of α-1,4-glucan chains, that is α-1,4-glucan chain extension,can proceed under amylosucrase-catalyzed transfer of α-D-glucosylradicals originating from sucrose. Suitable glucosyl group acceptorsare, in particular, short-chain and longer-chain oligo- andpolysaccharides having terminal glucose radicals which are linked viaα-1,4-glycosidic bonds. Preferably, the inventively used glucosyl groupacceptor is an unbranched, particularly preferably a branched, oligo- orpolysaccharide. Examples of inventive glucosyl group acceptors aremaltooligosaccharides such as maltopentaose, maltohexaose ormaltoheptaose.

Preferred glucosyl group acceptors are dextrins, amylopectins, amylosesand amylose-like polysaccharides, for example from corn and potatoes,and glycogens and glycogen-like polysaccharides, for example from muscletissue, mussels or bacteria.

Particularly preferred glucosyl group acceptors are branchedpolysaccharides such as glycogen. Such branched glucosyl group acceptorshave more than one end at which chain extension can take place. Thus theglycogen chain bears approximately 7-12% of branches to which glucosylradicals can be transferred.

Surprisingly, it has now been found that, in the amylosucrase-catalyzedsynthesis of α-1,4-glucans, polysaccharides having low polydispersitycan be obtained if the molar ratio of glucosyl group acceptor endsavailable for chain extension to sucrose and/or the weight ratio ofglucosyl group acceptor to sucrose in the reaction mixture assumes adefined minimum value. The chain extension reaction is presumed to bepreferred at this minimum value to side reactions which causepolysaccharide preparations of high polydispersity. The polydispersity,at a constant sucrose concentration, shows a tendency to decrease withincreasing concentration of the acceptor.

Expediently, the molar ratio of glucosyl group acceptor ends availablefor chain extension to sucrose in the reaction mixture is at least1:1000. Preferably, the molar ratio is at least 5:1000, and particularlypreferably at least 1:100. The upper limit for the molar ratio ofglucosyl group acceptor ends available for chain extension to sucrose isnot very critical and is expediently approximately 1:50 to 1:25.

The weight ratio of glucosyl group acceptor to sucrose is expediently atleast 1:50, for example at least 2:50, or at least 5:50. The optimumweight ratio depends on the type of acceptor. In the case of branchedpolysaccharide acceptors, the amount of acceptor required in thereaction mixture at a given sucrose concentration is generally lowerthan in the case of unbranched or only slightly branched polysaccharideacceptors. Thus, when glycogen having a weight-average molecular weightM_(w) of approximately 160 000 g/mol is used, a ratio of acceptor tosucrose of at least 2.5:50 has proved to be advantageous, while in thecase of dextrins having an M_(w) of approximately 5 000 to 6 000 g/mol,a weight ratio of acceptor to sucrose of at least 5:50 to 10:50 ispreferred.

For a given sucrose concentration and a given glucosyl group acceptor,the molecular weight of the polysaccharide obtained by the inventiveprocess is lower, the higher the concentration of glucosyl groupacceptor chosen. In this manner, by suitable choice of the weight ratioof glucosyl group acceptor to sucrose, the molecular weight of the endproduct can also be controlled.

The absolute concentration of the sucrose used as substrate of theamylosucrases in the reaction mixture is not critical. The amount used,however, expediently does not exceed 50% (w/v), since, above thisconcentration, the solution viscosity is too high and the reaction ratesharply decreases. Preferably, the sucrose concentration in the reactionmixture is between 1 and 30% (w/v).

The optimum conditions for the chain extension reaction, for examplemolar ratio of glucosyl group acceptor ends available for chainextension to sucrose, weight ratio of glucosyl group acceptor to sucroseand sucrose concentration in the reaction mixture, may be determinedwithout problem by simple experiments.

It has further been found that in the amylosucrase-catalyzed synthesisof α-1,4-glucans, polysaccharides having low polydispersity may beobtained when fructose is added to the reaction mixture. Presumably,addition of fructose inhibits interfering side reactions which give riseto polysaccharide preparations of high polydispersity. The effectintroduced by the presence of fructose is observed independently ofwhether the molar ratios and weight ratios of glucosyl group acceptor tosucrose have the above-specified minimum values or not. The addition offructose leads to a still narrower molecular weight distribution, thatis to a smaller polydispersity of the resultant end product, but inexchange the yield is somewhat lower.

Expediently, fructose is added to the reaction mixture at aconcentration of at least 10 mM. Preferably, the fructose is added at aconcentration of at least 50 mM, preferably 100 to 800 mM.

By means of the inventive process, an increase in the molecular weightof the glycosyl group acceptor used to twice to three times may beachieved, without problems. The molecular weight of the acceptor used inthe inventive process therefore also depends on the desired molecularweight of the end product. Since the reaction rate of the chainextension reaction increases with increasing degree of polymerization ofthe acceptor, expediently, however, acceptors having a weight-averagemolecular weight M_(w) of at least 0.5×10³ g/mol, preferably at least4×10³ g/mol, and particularly preferably at least 1×10⁵ to 1×10⁶ g/mol,are used. Since the polydispersity of the resultant reaction products isalso impaired by the uniformity of the acceptor material used, it isalso advisable to use acceptor molecules having the lowest possiblepolydispersity.

All enzymes can be used as amylosucrases which are able, according tothe reaction equation

Sucrose+(α-1,4-D-glucosyl)_(n)→D-fructose+(α-1,4-D-glucosyl)_(n+1)

to transfer the glucosyl radical of a sucrose molecule to the acceptormolecule with release of D-fructose and formation of an α-1,4-glucanchain. Preferably, amylosucrases from prokaryotes are used, inparticular from bacteria of the genus Neisseria. Suitable amylosucrasesare those occurring, for example, in the bacterial species N. sicca, N.canis, N. cinerea, N. perflava, N. subflava, N. dentrificans and N.polysaccharea. Preferably, amylosucrase from N. polysaccharea is used,for example from N. polysaccharea ATCC 43768.

The amylosucrases used can either be isolated directly from theorganisms in which they are naturally synthesized (MacKenzie, C. R. etal., Can. J. Microbiol., 24: 357-362; 1978), or, as described in WO95/31553, they can be produced by genetic engineering methods(recombinant amylo-sucrases). The enzymes can also be produced incell-free conditions using in vitro transcription and translationsystems.

The amylosucrases can be used not only as crude enzymes or in partiallypurified form, but also in highly purified form. Preferably, highlypurified amylosucrases are used, the term “highly purified amylosucrase”being taken to mean in particular an amylosucrase having a purity of atleast 80%, preferably at least 90%, and particularly preferably at least95%.

The use of highly purified amylosucrases in the inventive process hasthe advantage that the enzymes do not contain residues of the strain,for example the microorganism, from which they were isolated. Forexample, highly purified preparations do not contain other unwantedenzymes, for example polysaccharide-degrading enzymes such as amylases.The use of highly purified amylosucrases is also advantageous for use inthe food industry and pharmaceutical industry, since a reaction mediumwhich is defined and free from unnecessary constituents also gives amore precisely defined product. This leads to less complex authorizationprocesses for these biotechnologically produced products in the foodindustry and pharmaceutical industry, in particular if these productsare not to have any traces of transgenic microorganisms.

Preferably, recombinant amylosucrases are used as described, forexample, in WO 95/31553. Such recombinant amylosucrases can begenetically modified with respect to the naturally occurringamylosucrases, if appropriate also by mutations, for example insertions,deletions and substitutions, in order to modify defined properties ofthe expressed protein. Thus, the amylosucrase can, for example, beexpressed as a fusion protein together with a polypeptide sequence whosespecific binding properties enable easier isolation of the fusionprotein, for example by affinity chromatography (see, for example, Hoppet al., Bio/Technology 6 (1988), 1204-1210; Sassenfeld, TrendsBiotechnol. 8 (1980), 88-93). Particularly preferably, amylosucrases areused which are secreted by the host cells into the nutrient medium, sothat cell digestion and further purification of the enzyme are notnecessary, because the secreted enzyme can be obtained from thesupernatant. The amylosucrase can, as in the case of N. polysaccharea besecreted naturally, or secretion can be achieved by the enzyme beingexpressed together with a signal peptide, with the aid of which theenzyme can pass through the cell membrane of the host organism.

The amylosucrase can be used in free form or immobilized to a supportmaterial. Immobilization of the amylosucrase offers the advantage thatthe enzyme can be recovered in a simple manner from the reaction mediumand used repeatedly. Since the purification of enzymes is generallycostly and time-consuming, immobilization and reuse of the enzymepermits considerable cost savings. A further advantage is the purity ofthe reaction products, which contain no protein residues. Suitablesupport materials are, for example, agarose, alginate, cellulose,polyacrylamide, silica or nylon, with the coupling to the supportmaterial being via covalent or noncovalent bonds.

The amount of amylosucrase used is usually between 0.1 and 100 U/ml,preferably between 1 and 50 U/ml, and particularly preferably between 2and 25 U/ml.

The inventive polysaccharides are expediently prepared in vitro inbuffer-free or buffered aqueous systems having a pH between 4 and 9,preferably between 5.5 and 7.5. Suitable buffer systems are, forexample, citrate buffer, maleate buffer and acetate buffer.

The reaction temperature is expediently between 10 and 60° C.,preferably between 25 and 45° C.

The reaction is expediently carried out up to complete conversion of thesucrose. Usually, the reaction time for this is between 1 and 150 hours,for example between 10 and 100 hours.

The inventively formed polysaccharides are frequently sparingly solublein water and may therefore be separated off from the reaction mixturewithout difficulty, for example by centrifugation. Water-soluble orpartially water-soluble polysaccharides can be isolated, for example, byprecipitation with ethanol or by freezing out.

The inventive processes permit a simple and inexpensive preparation ofα-1,4-glucan-chain-containing polysaccharides of low polydispersity. Theprocesses are distinguished by an easy controllability of the molecularweight of the end products by an outstanding reproducibility. This makesit possible to prepare products of constant uniformity and purity andtherefore of high quality, which is of great importance for furtherindustrial use. The resultant products may be worked up inexpensively,since the process parameters which are required for the work up do notneed to be optimized anew for each work up batch.

DESCRIPTION OF THE DRAWINGS

The inventive process is useful in controlling the molecular weightduring the preparation of polysaccharides. In one embodiment, theinventive α-1,4-glucan-chain-containing polysaccharides are useful astablet fillers.

FIG. 1 shows the molecular mass distribution ofα-1,4-glucan-chain-containing polysaccharides in the reaction ofglycogen as glucosyl group acceptor with sucrose in the presence ofamylosucrase as a function of the glycogen concentration and sucroseconcentration.

FIG. 2a shows the molar mass distribution ofα-1,4-glucan-chain-containing polysaccharides in the reaction ofglycogen with sucrose in the presence of amylosucrase as a function ofthe glycogen concentration and sucrose concentration in the presence andabsence of fructose.

FIG. 2b shows the molar mass distribution ofα-1,4-glucan-chain-containing polysaccharides in the reaction of dextrinwith sucrose in the presence of amylosucrase as a function of dextrinconcentration and sucrose concentration in the presence and absence offructose.

The present invention is described in more detail by the examples below.

EXAMPLE 1

Purification of Amylosucrase

To produce amylosucrase, E. coli cells were used which had beentransformed with the vector pNB2 containing an amylosucrase fromNeisseria polysaccharea (WO 95/31553).

An overnight culture of these E. coli cells which secrete theamylosucrase from Neisseria polysaccharea was centrifuged and the cellswere resuspended in approximately 1/20 of the volume of 50 mM sodiumcitrate buffer (pH 6.5), 10 mM DTT (dithiothreitol), 1 mM PMSF(phenylmethyl-sulfonyl fluoride). The cells were then disintegratedtwice using a French press at 16 000 psi. Then 1 mM MgCl₂ and benzonase(Merck; 100 000 units; 250 units μl⁻¹) at a final concentration of 12.5units ml¹ were added to the cell extract. The mixture was then incubatedfor at least 30 min with gentle stirring at 37° C. The extract wasallowed to stand on ice for at least 1.5 hours. It was then centrifugedfor 30 min at approximately 40 000 g until the supernatant wasrelatively clear. Prefiltration via a PVDF membrane (Millipore“Durapore”, or similar) having a pore diameter of 0.45 μm was carriedout. The extract was allowed to stand overnight at 4° C. To carry outhydrophobic interaction (HI) chromatography, solid NaCl was added to theextract and a concentration of 2 M NaCl was set. The mixture was againcentrifuged for 30 min at 4° C. and approximately 40 000 g. The extractwas then freed from the final residues of E. coli by filtering itthrough a PVDF membrane (Millipore “Durapore” or similar) which had apore diameter of 0.22 μm. The filtered extract was separated on abutylsepharose-4B column (Pharmacia) (column volume: 93 ml, length: 17.5cm). Approximately 50 ml of extract having an amylosucrase activity of 1to 5 units⁻¹ were added to the column. Non-binding proteins were thenwashed from the column with 150 ml of buffer B (buffer B; 50 mM sodiumcitrate pH 6.5, 2 M NaCl). The amylosucrase was then eluted using adecreasing linear NaCl gradient (from 2 M to 0 M NaCl in 50 mM sodiumcitrate in a volume of 433 ml at a flow rate of 1.5 ml min⁻¹), which wasgenerated using an automatic pump system (FPLC, Pharmacia). Theamylosucrase was eluted between 0.7 M and 0.1 M NaCl. The fractions werecollected, desalted via a PD10-Sephadex column (Pharmacia), stabilizedwith 8.7% glycerol, tested for amylosucrase activity and then frozen instorage buffer (8.7% glycerol, 50 mM citrate).

EXAMPLE 2

Determination of Amylosucrase Activity

Purified protein or crude protein extract was added at various dilutionsto 1 ml assay solutions containing 5% sucrose, 0.1% glycogen and 100 mMcitrate, pH 6.5, and incubated at 37° C. After 5 min, 10 min, 15 min, 20min, 25 min, and 30 min, 100 μl were removed from this assay solutioneach time and the amylosucrase enzymatic activity was stopped by[imMediate] immediate heating for 10 min at 95° C. Using a coupledenzyme test, the amount of fructose released from sucrose by theamylosucrase was determined photometrically (M. Stitt et al., Methods inEnzymology 174:518-552; (1989). For this, 1 μl to 10 μl of theinactivated sample are added to 1 ml of 50 mM imidazole buffer pH 6.9, 2mM MgCl₂, 1 mM ATP, 0.4 mM NAD and 0.5 U/ml of hexokinase. Aftersequential addition of glucose-6-phosphate dehydrogenase (fromLeukonostoc mesenteroides) and phosphoglucose isomerase, the change inabsorption at 340 nm was measured. The amount of glucose released wasthen calculated using the Lambert-Beer law. If the result obtained isrelated to the time point of sampling, the number of enzyme units U maybe determined.

1U was defined as the amount of amylosucrase which, under theabovementioned conditions, releases 1 μmol of fructose/min.

EXAMPLE 3

Preparation of Polysaccharides

3.1 To prepare polysaccharide preparations, amylosucrase in 10 ml of 0.1M sodium acetate buffer, pH 6.5, 0.02% sodium azide was incubated at 37°C. with various concentrations of glycogen (Merck; M_(w) 160 000,polydispersity approximately 1.4) as glucosyl group acceptor and sucroseas substrate up to complete conversion of the sucrose, i.e. at least 48hr. The amylosucrase was added at a concentration of 5 to 20 U/ml. Aparallel control sample without glycogen was treated under otherwiseidentical conditions.

The polysaccharide precipitated out was centrifuged off (15 min, 1200 g)and washed twice by resuspension in water and repeated centrifugation.The pellets were frozen at −20° C. and freeze-dried at 0.34 mbar and anambient temperature of 25° C. (Alpha 1.4 freeze drier, Christ). Thesample temperature during drying was −25° C.

The resultant products were analyzed by gel permeation chromatography(GPC).

The operations were carried out as specified in DIN 55672-1. Allmeasurements were carried out in dimethyl sulfoxide (DMSO) using 0.09 MNaNO₃ as eluent. The GPC used a column combination of PS gel columns(10³, 10⁵ and 10⁶ Å; from PSS, Mainz, type “SDV 10 μ”). For detection ofthe mass fractions, a differential refractometer from Shodex, type “RI71” was used. A pump from Bischoff, type “HPLC Compact Pump” was used.The flow rate was 1 ml/min. Linear pullulans from PSS, Mainz, were usedfor calibration. (Since the samples had branched structures, themeasured results are not absolute but relative sizes, which, however,are comparable within a constant degree of branching of the samples.)The GPC software from PSS “Win-GPC scientific 4.02” was completely incompliance with DIN 55672-1 and was fully validated. The correctness ofthe data processing steps was therefore reproducible independently ofthe system. Molar masses having values less than 1000 g/mol were nottaken into consideration in the evaluation.

The results are listed in the table below and are shown graphically inFIG. 1 (w_(i) and W_(l) designate the normalized and relative massfractions of the ith polymer fraction). The perpendicular dotted lineshows the initial molecular weight of the glycogen used as glucosylgroup acceptor.

The results show that the polydispersity of the resultantpolysaccharides decreases drastically with constant sucroseconcentration and increasing glycogen concentrations.

TABLE Sucrose (%) Glycogen 5 10 20 40 mg/ml M_(w) M_(n) I_(n) M_(w)M_(n) I_(n) M_(w) M_(n) I_(n) M_(w) M_(n) I_(n) 0 67 170 2 129 32 7 2481 953 3.7 4 210 1 215 3.5 2 082 9 24 2.3 1 224 400 17 460 13 101 700 7894 13 11 250 2 811 4 2.5 251 400 28 080 9 95 670 4 701 20 5 315 300 71650 4.4 248 500 24 785 10.7 113 300 5 580 21.5 10 303 500 235 600 1.3316 900 76 380 4.1 247 900 12 380 20 200 700 5 069 40 20 244 100 195 8001.3 317 000 243 600 1.3 319 400 40 580 7.9 277 800 20 090 14 M_(w)Weight-average molecular weight M_(n) Number-average molecular weightI_(n) Polydispersity (M_(w)/M_(n))

3.2 In a further experiment, amylosucrase (5 U/ml) in 10 ml of 0.1 Msodium acetate buffer, pH 6.5, 0.02% sodium azide, 5% sucrose (w/v) wasincubated at 37° C. with glycogen at various concentrations and in thepresence of fructose (initial concentration 400 mM) or without fructoseto complete conversion of the sucrose (at least 48 hr). A parallelcontrol sample without enzyme was treated under otherwise identicalconditions. The resultant polysaccharide products were centrifuged offas described under 3.1, washed, freeze-dried and analyzed by GPC. Theresults are shown graphically in FIG. 2a, in which W_(i) has the meaningmentioned above. The vertical dotted line shows the initial molecularweight of the glycogen used as glucosyl group acceptor.

The results show that the polydispersity of the resultantpolysaccharides decreases with constant sucrose concentration andincreasing glycogen concentration. In the presence of fructose, afurther decrease in polydispersity is observed.

3.3 The experiment described under 3.2 was repeated under identicalconditions, but instead of glycogen, dextrin (Sigma No. D-4894, type IVfrom potatoes, M_(w) 6 650) was used. After incubation at 37° C. tocomplete conversion of sucrose (at least 48 hr) in the presence andabsence of fructose, the resultant polysaccharides were centrifuged offas described above, washed and freeze-dried. A parallel control samplewithout enzyme was treated under otherwise identical conditions andworked up and analyzed as described above. The results are showngraphically in FIG. 2, in which W_(l) has the meaning mentioned above.The vertical dotted line shows the initial molecular weight of thedextrin used as glucosyl group acceptor.

The results show that the polydispersity of the resultantpolysaccharides decreases with constant sucrose concentration andincreasing dextrin concentrations. In the presence of fructose, afurther decrease in polydispersity is observed.

What is claimed is:
 1. A process for preparingα-1,4-glucan-chain-containing polysaccharides, comprising the step ofsubjecting a glucosyl group acceptor to a chain extension reaction byreacting said glucosyl group acceptor with sucrose in the presence of anamylosucrase and with addition of fructose in a reaction mixture, saidfructose being added to the reaction mixture at a concentration of 100mM to 800 mM, the amount of glucosyl group acceptor in the reactionmixture being selected such that the molar ratio of glucosyl groupacceptor ends available for chain extension to sucrose is at least1:1000 and/or the weight ratio of glucosyl group acceptor to sucrose isat least 1:50.
 2. The process of claim 1, wherein the molar ratio ofglucosyl group acceptor ends available for chain extension to sucrose isat least 5:1000.
 3. A process for preparingα-1,4-glucan-chain-containing polysaccharides, which comprisessubjecting a glucosyl group acceptor to a chain extension reaction byreaction with sucrose in the presence of an amylosucrase and withaddition of fructose in a reaction mixture, wherein the fructose isadded to the reaction mixture at a concentration of 100 mM to 800 mM. 4.The process of claim 3, wherein the amylosucrase used is an amylosucrasefrom bacteria of the genus Neisseria.
 5. The process as claimed in claim4, wherein the amylosucrase used is an amylosucrase from bacteria of thespecies Neisseria polysaccharea.
 6. The process of claim 3, wherein theglucosyl group acceptor used is selected from the group consisting ofdextrins, amylose, amylopectin and glycogen.
 7. The process of claim 1,wherein the molar ratio of glucosyl group acceptor ends available forchain extension to sucrose is at least 1:100.
 8. The process of claim 1,wherein the amylosucrase used is an amylosucrase from bacteria of thegenus Neisseria.
 9. The process of claim 8, wherein the amylosucraseused is an amylosucrase from bacteria of the species Neisseriapolysaccharea.
 10. The process of claim 1, wherein the glucosyl groupacceptor used is selected from the group consisting of dextrins,amylose, amylopectin and glycogen.